Page 27 - Read Online

Li et al. Hepatoma Res. 2025;11:25 https://dx.doi.org/10.20517/2394-5079.2025.63 Page 5 of 17

[25]
highlighting its role in survival under stress . This stress activates the phosphatidylinositol 3-kinase
(PI3K)/mechanistic target of rapamycin (mTOR)/protein kinase B (AKT) pathway (regulates cell growth
and survival), enhancing HCC cell proliferation and invasion, whereas autophagy inhibitors can reduce
[26]
residual tumor growth after thermal stress . Hypoxia may synergize with autophagy; high microtubule-
associated protein 1A/1B-light chain 3B (LC3B; autophagosome marker) protein levels correlate with
[27]
elevated hypoxia-inducible factor 1α (HIF-1α) in tumor regions . Xu et al. found that silencing BCL2
interacting protein 3 (BNIP3; mediates mitophagy under hypoxia), a HIF-1α target, decreased heat-induced
[28]
LC3B and HIF-1α, supporting this link . Additionally, Yu et al. revealed that residual tumor cells post-
insufficient RFA (iRFA) recur through the HIF-1α/LC3B/sequestosome 1/p62 protein (P62) autophagy
pathway . Another key autophagy-related molecule, Sequestosome 1 (SQSTM1; encodes p62, a selective
[29]
autophagy receptor), has also been shown to be significantly associated with tumor recurrence after RFA .
[7]
Targeting the autophagy pathway may be an effective strategy to reduce recurrence.

EMT
EMT is essential for embryogenesis, wound healing, and malignant progression. In the context of neoplasia,
EMT enhances tumor-initiating and metastatic potential, as well as therapy resistance. Yoshida et al.
demonstrated that sublethal thermal treatment induces EMT in HCC cells, promoting a stem cell-like
phenotype with high proliferation . EMT transcription factors, including Snail (Snail family zinc finger
[8]
transcription factor - a master regulator that initiates EMT by repressing epithelial genes such as E-cadherin),
were notably upregulated by day 5 post-heat exposure . In post-ablation recurrent HCC specimens, cluster
[8]
of differentiation 44 (CD44), transforming growth factor-beta (TGF-β), twist family bHLH transcription
factor (TWIST), and Snail expression levels increased, strongly correlating with EMT activation. EMT
driven by protein kinase B (PKB, also known as Akt) and Extracellular signal-Regulated Kinase (ERK)
signaling reduces E-cadherin and increases Neuronal-cadherin (N-cadherin), matrix metalloproteinase-2
(MMP-2), and matrix metalloproteinase-9 (MMP-9), heightening lung metastasis risk .
[30]

Accumulation of β-catenin, a key wingless-related integration site (Wnt) factor, is a major mechanism
driving EMT after ablation. EMT correlates with nuclear β-catenin buildup, and its inhibition can reduce
invasiveness . Elevated Flotillin expression further enhances invasiveness via the Akt/Wnt/β-catenin
[31]
[32]
pathway . Additionally, under sublethal thermal stress, methyltransferase 1 (METTL1) and N7-
methylguanosine-modified transfer RNA (m7G tRNA) modifications enhance SLUG (Snail family
transcriptional repressor 2, encoded by the SNAI2 gene)/Snail translation in a codon frequency-dependent
[33]
manner . Blocking EMT-related proteins and pathways thus presents a promising strategy to reduce
recurrence and invasiveness of residual HCC cells post-ablation.

Hypoxic microenvironment formation
Thermal ablation induces tumor necrosis through high temperatures, but the resulting heat damage and
vascular occlusion create a hypoxic environment that supports residual tumor cell survival . Hypoxia-
[34]
inducible factors (HIFs), key transcription factors in heat and hypoxia response, accelerate residual cell
growth through mechanisms such as metabolic reprogramming, EMT, and angiogenesis . Studies show
[35]
that sublethal thermal stress-induced O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) stabilizes
HIF-1α, promoting the Warburg effect and tumor progression in HCC. Hypoxia also elevates TGF-β
expression, linked to malignant progression; in post-RFA tumor tissue, TGF-β levels are notably higher,
enhancing cancer stem cell (CSC) invasiveness via the HIF-1α/TGF-β1/Snail pathway [30,36] . Increased TGF-β
[37]
also induces myeloid-derived suppressor cells (MDSCs), fostering immunosuppression . Blocking TGF-β
signaling inhibits iRFA-induced progression and enhances anti-programmed death-1 (PD-1) antibody
efficacy . Hypoxia post-ablation raises vascular endothelial growth factor (VEGF) expression and
[38]
microvessel density. Kong et al. found that thermal treatment in human hepatocellular carcinoma cell line